1. Field of the Invention
The subject of the present invention is DNA fragments of Neisseria meningitidis which encodes the transferrin receptor subunits as well as a process for producing each of the subunits by the recombinant route.
Meningitides are generally either of viral origin or of bacterial origin. The bacteria mainly responsible are: N. meningitidis and Haemophilus influenzae, which are respectively implicated in about 40 and 50% of cases of bacterial meningitides.
In France, about 600 to 800 cases of N. meningitidismeningitides are recorded per year. In the United States, the number of cases is up to about 2,500 to 3,000 per year.
The N. meningitidis species is sub-divided into serogroups according to the nature of the capsular polysaccharides. Although about twelve serogroups exist, 90% of meningitis cases can be attributed to 3 serogroups: A, B and C.
2. Description of the Related Art
Effective vaccines based on capsular polysaccharides exist for the prevention of meningitides caused by N. meningitidis serogroups A and C. These polysaccharides as they are only slightly or not at all immunogenic in children below 2 years and do not induce immunological memory. However, these disadvantages can be overcome by conjugating these polysaccharides with a carrier protein.
In contrast, the polysaccharide of N. meningitidis group B is not at all or is only slightly immunogenic in man whether it is in conjugated form or not. Thus it appears highly desirable to seek out a vaccine against meningitides induced by N. meningitidis especially of the serogroup B other than a polysaccharide-based vaccine.
To this end, various proteins of the outer membrane of N. meningitidis have already been proposed. These are in particular the membrane receptor for human transferrin.
In general, the great majority of bacteria require iron for their growth and they have developed specific systems for acquiring this metal. With regard especially to N. meningitidis which is a strict pathogen for man, the iron can only be derived from human iron transport proteins such as transferrin and lactoferrin since the quantity of iron in free form is negligible in man (of the order of 10.sup.-18 M), in any case insufficient to permit bacterial growth.
Thus, N. meningitidis has a human transferrin receptor and a human lactoferrin receptor which enable it to bind these iron-chelating proteins and subsequently to capture the iron required for its growth.
The transferrin receptor of the strain B16B6 of N. meningitidis has been purified by Schryvers et al. (WO 90/12591) from a membrane extract. This protein, as purified, appears to consist essentially of 2 types of polypeptides: a polypeptide with a high apparent molecular weight of 100 kD and a polypeptide with a lower apparent molecular weight of about 70 kD, as visualised after SDS-polyacrylamide gel electrophoresis.
The purification product especially identified by Schryvers is by arbitrary definition and for the purposes of the present patent application, called transferrin receptor and its constituent polypeptides, subunits. In the text which follows, the subunits of high molecular weight and of lower molecular weight are called Tbp1 and Tbp2 respectively.
However, the purification process described by Schryvers et al. cannot be used for the large-scale production of the transferrin receptor. The industrial preparation of this receptor in purified form necessarily involves a production step using a heterologous expression system.